Dissertation on
A STUDY OF THYROID DYSFUNCTION AND ITS ASSOCIATED RISK FACTORS AMONG TYPE 2
DIABETES MELLITUS PATIENTS
Submitted to
THE TAMILNADU Dr. M. G. R. MEDICAL UNIVERSITY Chennai – 600032
In partial fulfilment of the requirement for the award of degree of
DOCTOR OF MEDICINE IN BIOCHEMISTRY BRANCH –XIII
Submitted by
Register number : 201523352
KARPAGA VINAYAGA INSTITUTE OF MEDICAL SCIENCES AND RESEARCH CENTRE
MADHURANTHAGAM TAMILNADU
MAY 2018
CERTIFICATION
This is to certify that “A Study of Thyroid Dysfunction and its associated risk factors among Type 2 Diabetes Mellitus patients” is a bonafide work of Dr.C.Sathishkumar in partial fulfilment of the requirements for the M.D Biochemistry (Branch XIII) examination of The Tamilnadu Dr.M.G.R Medical University to be held on May 2018.
Dr. Aruna kumari. R., MD Dr. Sufala Sunil Viswas Rao., MD Head of Department, Principal,
Professor and Guide, Karpaga Vinayaga Institute Department of Biochemistry, of Medical Sciences,
Karpaga Vinayaga Institute Madhuranthagam.
of Medical Sciences, Madhuranthagam.
CERTIFICATION
This is to certify that “A Study of Thyroid Dysfunction and its associated risk factors among Type 2 Diabetes Mellitus patients” is a bonafide work of Dr.C.Sathishkumar in partial fulfilment of the requirements for the M.D Biochemistry (Branch XIII) examination of The Tamilnadu Dr.M.G.R Medical University to be held on May 2018.
Dr.Aruna kumari. R MD., Head of Department, Professor and Guide,
Department of Biochemistry, Karpaga Vinayaga Institute of Medical Sciences,
Madhuranthagam.
DECLARATION
I, Dr.C.Sathishkumar hereby declare that this dissertation “A Study of Thyroid Dysfunction and its associated risk factors among Type 2 Diabetes Mellitus patients” is a presentation of my own work and that it has not been submitted anywhere for any award.
Wherever contributions of others are involved, every effort is made to indicate this clearly, with due reference to literature and discussions.
This work was done under the guidance of Professor Dr.Aruna kumari.R MD, at Karpaga Vinayaga Institute of Medical Sciences, Madhuranthagam.
Candidate’s Name: Dr. C. Sathishkumar
Candidate’s signature:
Date:
In the capacity as guide for the candidate’s dissertation work, I certified that the above statements are true to the best of my knowledge.
Dr.Aruna kumari.,MD Head of Department, Professor and Guide, Department of Biochemistry, Karpaga Vinayaga Institute of Medical Sciences,
Madhuranthagam.
ACKNOWLEDGEMENT
I bow myself in front of the Almighty expressing my thankfulness for all his blessings throughout my life.
I express my sincere and heartfelt gratitude to our respected Managing Director, Professor Dr.R.Annamalai., M.S., for permitting and for extending his valuable support in conducting this study.
I humbly respect and thank our former Dean, Professor Dr.A.R.Chakaravarthy., M.S., and Principal Dr.Sufala Sunil Viswas Rao., M.D., for permitting and supporting me to conduct this study.
“There are two kinds of teachers; the kind that fill you with so much quail shot that you can’t move, and the kind that just gives you a little prod behind and you jump to the skies.” - Robert Frost
By God’s grace, I am blessed to be a student of a great teacher, who is always there to direct her students in the right direction, who does not put undue pressure and She is the one who taught the virtue of simplification and understanding in learning. She always greets us with a beautiful smile and shows that Knowledge is her power. She is none other than our esteemed and beloved, Head of Department of Biochemistry and my guide, Professor Dr.Aruna Kumari. R. MD. The word “Gratitude” falls short for her. I express my deep, heartfelt and sincere thanks to her for her guidance in every process of this study.
I would like to thanks my Professor Dr.Subiman Saha for his wisdom filled words. I would like to thank Dr.Khadeja Bi my Assistant Professor for her kind support and timely guidance for my study.
I would like to express my sincere gratitude to Dr.Siva, Ph.D., Associate Professor for his unflinching support and encouragement throughout this study.
I would like to thank Dr.Firoz Pathan., Assistant Professor for his kind support and encouraging words during the dissertation work.
I would like to thank Dr.Amar Nakeshkumar.,Ph.D., for his unbounded support and suggestions during the dissertation work.
I express my sincere thanks to Mr.Saravanan.,M.Sc.,Ph.D., Assistant Professor for his concern, affection, priceless support and willingness to help at all times.
I extend my immense thanks and gratitude to Dr.Selvakumar Kandaswamy, Ph.D., Clinical Biochemist, Kauvery Hospital, Chennai for his tremendous support and boundless guidance for the completion of dissertation work and for being constant source of inspiration.
I also wish to thank Mr.R.Radhakrishnan., Non-Medical Demonstrator for his kind support and guidance during the dissertation work and I would like to thank our Laboratory Technicians of Undergraduate Laboratory and Central Research Laboratory for their help and Co-operation
I would like to thanks Mrs.Gladius Jennifer for her guidance towards statistical analysis techniques.
I immensely thank my Co-PG’s for their help and great support during the course of the study.
Many thanks to all patients and volunteers who participated in the study.
Last but not the least; I am grateful to my beloved parents Mr.S.Chakravarthy & Mrs.N.Kanchana and all my family members for their unbounded, unconditional love, trust and support throughout my life and especially during my post graduation period. Because without their support this would not have been possible.
TABLE OF CONTENTS
S.
NO.
TITLE
PAGE NO.
1. INTRODUCTION
2. AIMS AND OBJECTIVES
3. REVIEW OF LITERATURE
4. MATERIALS AND METHODS
5. RESULTS
6. DISCUSSION
7. CONCLUSION
8. ANNEXURES
9. BIBLIOGRAPHY
ABBREVIATIONS
LIST OF ABBREVIATIONS
ACh Acetyl Choline
ADA American Diabetes Association
ATP Adenosine Tri Phosphate
BMI Body Mass Index
CHOD POD Cholesterol Oxidase Peroxidase
Cyclic AMP Cyclic Adenosine Mono Phosphate
GLUT-4 Glucose Transporter
HbA1C Glycosylated Hemoglobin
HDL High Density Lipoprotein
IGF-1 Insulin Growth Factor-1
IL-6 Interleukin-6
IR Insulin Receptor
IRS-1 gene Insulin Receptor Substrate – 1 gene
LDL Low Density Lipoprotein
NHANES III Study National Health And Nutrition Examination Survey III Study
TC Total Cholesterol
TD Thyroid Dysfunction
TGL Triglycerides
TNF-α Tumor Necrosis Factor-α
TRH Thyrotropin Releasing Hormone
TSH Thyroid Stimulating Hormone
VLDL Very Low Density Lipoprotein
WHO World Health Organization
INTRODUCTION
1
INTRODUCTION
Diabetes mellitus (DM) is a chronic metabolic disorder that results in hyperglycemia (high blood glucose levels) due to being ineffective at using the insulin it has formed; also known as insulin resistance and or being unable to synthesis enough insulin. In which occurrence has been steadily increasing throughout the world. DM is becoming a fast epidemic in some countries of the world with the number of people affected. This would expect to be double in the next decade due to increase in ageing population. It is also a one of the leading cause of death worldwide. [1]
Individuals existing with type 2 DM are more vulnerable to various forms of complications both short- and long-term, which often lead to their premature death. This tendency of increased morbidity and mortality is seen in patients with type 2 DM because of the commonness of this type of DM, its insidious onset and late recognition, especially in resource-poor developing countries like Africa. [2]
In 2014, the International Diabetes Federation estimated that 387 million people around the world had DM, and by 2035 this number is likely to rise to 592 million. Such factors as inactive lifestyle, dietary modifications, ethnicity, and obesity have led to a remarkable increase in the occurrence of DM, particularly in the twenty-first century.[3]
Diabetes mellitus (DM) is almost certainly one of the oldest diseases known to man. It was first reported in Egyptian manuscript about 3000 years ago.[4] In 1936, the difference between type 1 and type 2 DM was clearly
2
made. Type 2 DM was first described earlier as a factor of metabolic syndrome.[5] Type 2 DM (formerly known as non-insulin dependent DM) is the most common form of DM characterized by hyperglycemia, insulin resistance, and relative insulin deficiency.[6] Type 2 DM results from statement among genetic, environmental and behavioural hazard factors.[7]
In type 2 diabetes mellitus (T2DM) the primary defects observed are developed insulin resistance and abnormal insulin secretion by pancreatic beta-cells. DM is a common metabolic disorder considered by absolute or relative deficiencies in insulin secretion and/or insulin action associated with chronic hyperglycemia and conflict of carbohydrate, lipid and protein metabolism.
The thyroid gland is significantly concerned in metabolism of lipid and carbohydrate, role in metabolism of adipogenesis and thermogenesis, regulation of body weight.[8] Thyroid dysfunction may also affect and interfere in control of diabetes. Hyperthyroidism is characteristically associated with deteriorating glycemic control and amplified insulin requirements. There is primary increased hepatic gluconeogenesis, rapid gastrointestinal glucose absorption, and probably increased insulin resistance.
Indeed, thyrotoxicosis may unmask latent diabetes8. The most common thyroid disorder is hypothyroidism. The association of thyroid disorder with diabetes is more frequent in diabetics who have deranged metabolic control. Thyroid also influences the glycosylated haemoglobin levels.[9]
3
In practice, there are several implications for patients with both diabetes and hyperthyroidism. First, in hyperthyroid patients, the diagnosis of glucose intolerance needs to be considered cautiously, since the hyperglycemia may improve with treatment of thyrotoxicosis. Second, underlying hyperthyroidism must be considered in diabetic patients with unexplained worsening hyperglycemia. Third, clinician need to anticipate potential deterioration in glycemic control and amend the treatment accordingly in patients with diabetic and hyperthyroidism, lower the blood glucose level will found in euthyroidism restoration.[10]
Although wide-ranging changes in carbohydrate metabolism are seen in
hypothyroidism, clinical manifestation of these abnormalities is seldom conspicuous. However, the condensed rate of insulin degradation may inferior
the exogenous insulin condition. The presence of hypoglycemia is uncommon
in isolated thyroid hormone deficiency and should elevate the opportunity of hypopituitarism in a hypothyroid patient. More importantly, hypothyroidism is
accompanied by a variety of abnormalities in plasma lipid metabolism, including elevated triglyceride and low-density lipoprotein (LDL) cholesterol concentrations.[11] Even subclinical hypothyroidism can exacerbate the coexisting dyslipidemia commonly found in type 2 diabetes and further increase the risk of cardiovascular diseases. Lipid abnormalities can reverse by replacement of sufficient thyroxine.
Prevalence of thyroid dysfunction is higher in type 2 diabetes population compared to normal population. Diabetes mellitus and thyroid dysfunction are
4
the most common endocrine diseases seen among the adult population[11] while insulin or thyroid hormones metabolism can result in functional abnormalities of one another. The strong link between diabetes and thyroid diseases encouraged the American Diabetes Association (ADA) to propose that people with diabetes must be checked periodically for thyroid dysfunction.[12]
Thyroid hormones may influence glucose manage through a variety of actions on intermediary metabolism. One of these effects becomes clinically appropriate in patients with co-existent diabetes and hyperthyroidism. Excess thyroid hormones promote hyperglycaemia by facilitating increasing insulin clearance, glucose intestinal absorption, and glycogenolysis and gluconeogenesis enhancement. Also, hyperthyroidism is associated with increased hepatic glucose output, reduced insulin action and increased lipoly-
sis13 (Potenza et al., 2009). Accordingly, diabetic patients with overt hyperthyroidism may experience poor glycaemic control and indeed hyperthyroidism has been known to impulsive diabetic ketoacidosis in patients with diabetes.[14] Thyroid disease must be screened every year in diabetic patients to detect asymptomatic thyroid dysfunction.[15] At the same time, patients with thyroid dysfunction may require to be tested for the prospect of abnormal glucose metabolism, since extreme thyroid hormones cause increased glucose production in the liver, rapid absorption of glucose through the intestine and increased insulin resistance.[16] There are many risk factors known to be associated with thyroid dysfunction in the general population, including age, gender, BMI, family history of thyroid disease, smoking, and pregnancy.
5
Incidence of hyperthyroidism and hypothyroidism increases with age, especially beyond 30 years, and it has been established that female gender is 10–20 times more likely to have this medical problem than males.[17]
Morbidly accounting for 19.5% obese individuals show a high prevalence of overt and subclinical hypothyroidism.[ 8] Risk factors for thyroid dysfunction among diabetic patients are similar to what have been reported in non-diabetics, although they will vary with the type of thyroid dysfunction., while hypothyroidism among diabetic patients is more prevalent among women[18] and the older population.[19]
Thus, the present study was intended to explore a study of thyroid dysfunction and associated risk factors among type 2 diabetes mellitus patients in Tamil Nadu.
AIMS AND OBJECTIVES
6
AIM AND OBJECTIVES
Aim
To evaluate thyroid profile, lipid profile, and renal parameters in type 2 Diabetes mellitus
Objectives
To assess the risk factors like thyroid profile, lipid profile and renal
parameters for cardiovascular disease and renal disease in type 2 diabetes Mellitus among male and female patients
To compare the serum levels of thyroid profile, lipid profile and renal parameters between controlled and uncontrolled diabetes patients
REVIEW OF LITERATURE
7
REVIEW OF LITERATURE
THYROID GLAND
The thyroid gland is among the most significant organs of the endocrine system and has a weight of 15-20g. It is soft and its colour is red. This organ is located between the C5-T1 vertebrae of columna vertebralis, in front of the trachea and below the larynx. It is comprised of two lobes (lobus dexter and lobus sinister) and the isthmus that binds them together. Capsule glandular which is internal and external folium of thyroid gland is wrapped up by a fibrosis capsule named thyroid. The thyroid gland is nourished by a thyroidea superior that is the branch of a. carotis external and a. thyroid inferior that is the branch of a subclavia.[20-27]
THYROID HORMONES AND ITS FUNCTIONS
The thyroid hormones, triiodothyronine (T3) and its prohormone, thyroxine (T4), are tyrosine-based hormones produced by the thyroid gland that are primarily responsible for regulation of metabolism. T3 and T4 are partially composed of iodine (see molecular model). A deficiency of iodine leads to decreased production of T3 and T4, enlarges the thyroid tissue and will cause the disease known as simple goitre. T4 increase the spectacular apoptosis (programmed cell death) of the cells of the larval gills, tail and fins. Contrary to amphibian metamorphosis, hypothyroidism and thyroidectomy in mammals may be considered a sort of phylogenetic and metabolic regression to a forestage of reptilian life. Indeed, many disorders that seem to afflict hypothyroid humans have reptilian-like features, such as scaly , dry, hairless,
8
cold skin and a general slowing of metabolism, heart rate , digestion, and nervous reflexes, with lethargic cerebration, hyperuricemia and hypothermia.
[28]
IODINE
Iodine is taken into the body oral. Among the foods that contain iodine are seafood, iodine-rich vegetables grown in soil, and iodized salt. For this reason, iodine intake geographically differs in the world. Places that are seen predominantly to have iodine deficiency are icy mountainous areas and daily iodine intake in these places is less than 25 µg. Hence, diseases due to iodine deficiency are more common in these geographies. Cretinism in which mental retardation is significant was first identified in the Western Alps. [27, 29]
Iodine absorbed from the gastrointestinal system immediately diffuses in extra cellular fluid. T3 and T4 hormones are fundamentally formed by the addition of iodine to tyrosine amino acids. While the most synthesized hormone in thyroid gland is T4, the most efficient hormone is T3. [22, 24]
THYROGLOBULIN
The synthesized thyroglobulin is transported to the apical section of the cell and passes to the follicular lumen through exocytose, and then joins thyroid hormone synthesis [27, 29]. Through this deiodinization, about 50% of iodine in the thyroglobulin structure is taken back and can be reused. Iodine deficiency in individuals lacking this enzyme, and correspondingly,
9
hypothyroid goiter is observed. Such patients are given iodine replacement treatment. [26, 31]
ALBUMIN
Serum albumin is a protein with a molecule weight of 65kDa and has a lower rate of binding even though its plasma concentration is the highest. [22]
Synthesis and secretions need to be kept at a certain level in order for the liveliness of thyroid hormones to be maintained. In this respect, the most important mechanism in controlling the synthesis and secretion of thyroid hormones is the hypothalamus-hypophysis-thyroid axis. Another one is the auto control mechanism that is dependent on iodine concentration as noted earlier [26]
10 HORMONES
TRH also increases the secretions of growth hormone (GH), follicle stimulating hormone (FSH), and prolactin (PRL). While the TRH secretion is increased by noradrenaline, somatostatin and serotonin inhibits it. [26]
METABOLISM
Thyroid hormones carry out their metabolic effects by carbohydrates, fat and protein metabolisms, vitamins, basal metabolic rate and its effect on body weight. When the effects of thyroid hormones on carbohydrate metabolism are observed, it is established that it is both anabolic and catabolic. As a result of thyroid hormones increasing the enzyme synthesis due to protein synthesis in cells, enzymes in carbohydrate metabolism also increase their activities. Thus, thyroid hormones increase the entrance of glucose into the cell, absorption of glucose from the gastrointestinal system, both glycolysis and gluconeogenesis, and secondarily, insulin secretion [26, 29]
11
THYROID HORMONE METABOLISM
In addition, as metabolism products also increase due to an increase in oxygen consumption when thyroid hormones are over secreted, vasodilation occurs in periphery. Thus, blood flow increases, and cardiac output can be observed to be 60% more than normal. The thyroid hormone also raises the heart rate due to its direct increasing effect on heart stimulation.[21] Thyroid hormones increase the contraction of heart muscles only when they raise it in small amounts. When thyroid hormones are over secreted, a significant decrease occurs in muscle strength, and even myocardial infarction is observed in severely thyrotoxic patients. [26, 31]
12 TYPES OF GLAND
Endocrine Glands are the glands which contain no duct and release their secretions into the intercellular fluid or directly into the blood. The compilation of endocrine glands makes up the endocrine system.
1. The important endocrine glands are the pituitary (anterior and posterior lobes), thyroid, parathyroid, pancreas , adrenal (cortex and medulla), and gonads.
2. The thyroid gland comprises of two lateral masses, held by a cross bridge, that are fixed to the trachea. They are slightly lower in position to the larynx.
3. The parathyroid glands are four masses of tissue, two lodged posterior in each lateral mass of the thyroid gland.
4. One adrenal gland is situated on top of each kidney. The cortex is the outer most layer of the adrenal gland. The medulla is the inner core.
5. The pancreas is situated along the lower curvature of the stomach, close to where it meets the first part of the small intestine, the duodenum.
6. The ovaries and testes are found in the pelvic cavity.
HORMONES AND TYPES
The endocrine system secretes hormones that are important in maintaining regulation of reproduction , homeostasis and development. A hormone is a chemical messenger secreted by a cell that effects specific change in the cellular activity of the target cells. But exocrine glands which secrete
13
substances such as milk, saliva , stomach acid and digestive enzymes.
Endocrine glands do not secrete substances into ducts (tubes). Instead, they secrete their hormones directly into the surrounding extra cellular space. The hormones then enter into the nearby capillaries and then they are transported throughout the body in the blood.
Classification of Hormones based on chemical nature :
Amino acid-derived, Polypeptide and proteins, Steroids, Eicosanoids [22].
Lipid-soluble hormones (steroid hormones and hormones of the thyroid gland) enter through the cell membranes of target cells. The lipid-soluble hormone then attach to a protein receptor that, in turn, promotes a DNA segment which turns on specific genes. The proteins transcription in the genes and results in subsequent translation of mRNA which acts as enzymes that will regulate specific physiological actions of the cell.
Water-soluble hormones like polypeptide, protein, and most amino acid hormones , attach to a receptor protein on the plasma membrane of the cell [22]. The receptor protein, in turn, activates the production of second messengers which results in the synthesis and secretion of hormones to that specific tissue.
DIABETES
Diabetes Mellitus is a disorder caused by the total or relative absence of insulin, with or without insulin resistance that leads to dysregulation of carbohydrate , fat and protein metabolism which manifests clinically as an elevated blood glucose. Diabetes mellitus becoming the epidemic of the
14
twenty first century with more than 1.5 million deaths worldwide directly attribute to this disease in 2012 (WHO). Despite advances in medical treatment, cardiovascular disease (CVD) remains the important cause of morbidity and mortality in individuals with diabetes. The risk of vascular complications is double in diabetes mellitus resulting in significant reduction in life expectancy. [32]
After a vascular event occurred, the outcome in patients with diabetes mellitus is worse when compared with individuals with normal glucose metabolism, regardless of the therapeutic strategy used in the acute stage[33, 34,
35, 36, 37].
There are two important reasons for the adverse vascular outcome in patients with diabetes mellitus. The first one is related to more extensive vascular pathology and the second one involves an enhanced thrombotic environment. [38]
STRUCTURE OF INSULIN AND PROPERTIES
Insulin a polypeptide found in 1928 and sequence of amino acid identified in 1952. It is a dipeptide, containing A and B chains linked by disulphide bridges, and containing 51 amino acids, contains molecular weight of 5802. The isoelectric point is pH 5.5. [39] A chain consists of 21 amino acids and the B chain 30 amino acids. A chain has an N-terminal helix related to an anti-parallel C-terminal helix; the B chain has a central helical segment. These two chains are connected by 2 disulphide bonds, which link the N- and C-terminal helices of the A chain to the central helix of the B chain.[40]
15 SYNTHESIS AND RELEASE OF INSULIN
The short arm of chromosome 11[41]7 is coded for Insulin and synthesised in the β cells of the pancreatic islets of Langherhans as its precursor, proinsulin (PI). PI is synthesised in the Ribosomes of the rough endoplasmic reticulum from mRNA as pre-proinsulin (PPI). This PPI is produced by sequential synthesis of a signal peptide, B chain, the linking peptide and then the A chain comprising a single chain of 100 amino acids.
Secretory vesicles transfer proinsulin from the rough endoplasmic reticulum to the Golgi apparatus, which favours formation of soluble zinc-containing proinsulin hexamers using aqueous zinc and calcium rich environment.[40] As immature storage vesicles form from the Golgi, enzymes acting outside the Golgi convert proinsulin to insulin and C-peptide.[42] Insulin forms zinc- containing hexamers which are insoluble, precipitating as chemically stable crystals at pH 5.5. Granules are get matured and secreted into the blood stream by exocytosis, insulin, and an equal concentration ratio of C-peptide were also released. Proinsulin and zinc usually comprise no more than 6% of the islet cell secretion. [40]
Insulin secretion from the islet cells into the portal veins is pulsatile. An ultradian oscillatory mould of insulin discharge, in addition to post meal deviation, has been reported. [43] In response to a stimulus such as glucose, insulin secretion is characteristically biphasic, with an initial rapid phase of
16
insulin secretion, followed by a less intense but more sustained release of the hormone. [44]
FACTORS INFLUENCING BIOSYNTHESIS OF INSULIN AND ITS RELEASE
Insulin secretion may be subjective by alterations in production at the level of gene transcription, translation, and post-translational modification in the Golgi as well as by factors influencing insulin release from secretory granules. Longer-term modification may occur via influences on β cell mass and differentiation. [45] Insulin plays a pivotal role in glucose consumption and metabolism, it is not unanticipated that glucose has numerous influences on insulin biosynthesis and secretion. However, other factors such as fatty acids, amino acids, Ach, glucagon-like peptide-1 (GLP-1), and several other agonists, together in arrangement, also influence this processes. [44]
MOLECULAR MECHANISMS OF SECRETION OF INSULIN
Increased levels of glucose provoke the initial phase of glucose- mediated insulin secretion by release of insulin from β cell. Glucose entry into the β cell is sensed by glucokinase, which phosphorylates glucose to glucose-6- phosphate (G6P), generating ATP.12 Closure of K+-ATP-dependent channels results in membrane depolarization and commencement of voltage dependent calcium channels leading to an raise in intracellular calcium concentration;
which triggers insulin secretion in a pulsatile manner. [46] Escalation of this
17
reaction occurs by both a potassium-ATP channel-independent Calcium dependent pathway and potassium ATP channel-independent calcium self- directed pathways of glucose action. [44] Other intermediaries are activation of phospholipases and protein kinase C (i.e., acetycholine) and by stimulus of adenylyl cyclase action and commencement of β cell protein kinase A, which potentiates insulin secretion. This latter mechanism may be activated by hormones and appear to partake an important role in the second part of glucose mediated insulin secretion, granules are translocated from reserve pools which is responsible for the refilling of insulin. [44]
REGULATION OF INSULIN SECRETION
Nutrient and non-nutrient secretagogues play a vital role in regulation of synthesis and secretion of insulin, in the context of stimuli from environmental and the interaction of other hormones. [42] Nutrient secretagogues such as glucose appear to generate insulin secretion from the β cell by increasing intracellular ATP and closing of potassium ATP channels as described above.
Generation of cyclic AMP and other cellular energy intermediates are also amplified, further enhancing insulin release. Glucose does not require insulin action to enter the β cell [42] Non-nutrient secretagogues might act via neural stimuli such as cholinergic and adrenergic pathways, or through peptide hormones.
18
INSULIN SECRETION AND ITS PHYSIOLOGY
Glucose is the principal stimulus for insulin secretion, although other hormones, macronutrients and neural input may adjust this response. Insulin, together with its principal counter-regulatory hormone glucagon, regulates blood glucose concentrations. [47] β cells secreted from pancreas is 0.25–1.5 units of insulin/ hour during the basal (or fasting) state, enough to allow glucose insulin-dependent access into cells. This concentration prevents unconstrained hydrolysis of triglycerides and controlled gluconeogenesis, thus maintaining normal fasting glucose levels. Insulin basal state secretion accounts above for 50% of total 24 hour insulin secretion. Successive insulin secretion into the portal venous system, 60% is consequently separated by the liver; so portal vein insulin concentrations attain the liver approach triple that of the peripheral circulation. Circulating fasting insulin concentrations in healthy individuals are about 3–15 mIU/L [47]
INSULIN SECRETION IN RESPONSES TO GLUCOSE
An individual in healthy condition, glucose activates biphasic pancreatic secretion. Intravenous administration of glucose is related with a instantaneous
“primary phase” of insulin release within 1 minute, peaking at 3–5 minutes, and lasting about 10 minutes; the slower beginning “second phase” of insulin secretion begins soon after the glucose bolus but is not obvious until 10 minutes later, lasts the period of the hyperglycaemia and is comparative to the glucose absorption immediately prior to the glucose administration. [47]
19
The primary phase of insulin secretion represents release of insulin previously synthesised and stored in secretory granules; the second phase represents newly synthesised insulin and insulin secretion stored. On the whole insulin secretion relates to the entire dose of glucose and its pace of administration; occurs with 20 g of glucose given intravenously over 3 minutes in humans is the maximal pancreatic response. [48]
In compare to the reproducible pattern of insulin secretion in response to intravenous glucose, insulin secretion following oral glucose is greatly more variable. With an oral glucose load, gastric emptying and gastrointestinal motility affect glucose absorption, gastro-intestinal hormones and neural input linked with glucose ingestion adjust the insulin response, and insulin secretion continues some time after glucose intake. [47]
MECHANISMS OF INSULIN RESISTANCE
Physiologically, the entire body levels, the actions of insulin are prejudiced by the interaction of other hormones. Insulin, even if the dominant hormone driving metabolic processes in the fed state, acts in performance with growth hormone and Insulin growth factor-1; growth hormone is secreted in reaction to insulin, among other stimuli, preventing insulin-induced hypoglycaemia. Other counter-regulatory hormones comprise glucagon, glucocorticoids and catecholamines. These hormones drive metabolic processes in the fasting state. Glucagon promotes ketogenesis gluconeogenesis and glycogenolysis. The relative amount of insulin to glucagons determines the
20
quantity of phosphorylation or dephosphorylation of the relevant enzymes.[49]
Catecholamines promotes glucocorticoids, lipolysis and glycogenolysis;
promote catabolism of the muscle, gluconeogenesis and lipolysis. Surplus secretion of these hormones may donate to insulin resistance in meticulous settings, but does not description for the vast mainstream of insulin resistant states.
Post-receptor defects in insulin signalling initiate the insulin resistance in most cases which is believed to be manifest at the cellular level. Even though promising result in experimental animals with respect to a variety of insulin signalling defects, their significance to human insulin resistance is currently unclear. Potential mechanisms consist of down-regulation, deficiencies or genetic polymorphisms of tyrosine phosphorylation of the IR, IRS proteins or PIP-3 kinase, or may involve abnormality of GLUT 4 utility.
[50]
INSULIN RESISTANCE AND THE SITES OF INSULIN ACTION
Insulin, insulin deficiency and insulin resistance effects may vary according to the physiological role of the organs and tissues concerned, and their dependence on metabolic processes of insulin. Those tissues definite as insulin dependent, based on glucose transport in intracellular, are predominantly adipose tissue and muscle. However, actions of insulin are pleotropic and extensive, as are the manifestations of insulin resistance and the hyperinsulinaemia as associated compensatory. [51]
21 Adipose Tissue
Glucose transport into intracellular adipocytes in the postprandial state is insulin-dependent GLUT 4; it is estimated that accounts for about 10% of insulin stimulated among whole body glucose uptake is adipose tissue.34 Insulin stimulates glucose uptake, promotes lipogenesis while suppressing lipolysis, and consequently free fatty acid fluctuation into the bloodstream. As adipocytes are not reliant on glucose in the basal state, intracellular energy may be supplied by fatty acid oxidation takes place in insulin-deficient states, whereas liberating free fatty acids into the circulation for undeviating utilization by other organs e.g. heart, or liver where they are transformed to ketone bodies. During prolonged starvation these ketone bodies provide an alternative energy substrate for the brain. [52]
Muscle
GLUT 4 is essential for insulin dependent glucose uptake into muscle is, and 60–70% of whole-body insulin mediated uptake is take part in muscle. [53]
In the fed condition insulin promotes glycogen synthesis via glycogen synthase activation. This enables energy to be unconfined anaerobically via glycolysis, e.g. during intense muscular action. Muscle cells do not depend on glycogen (or glucose) for energy during the fasting state, when insulin levels are low.
Insulin releases amino acids for gluconeogenesis and suppresses protein catabolism while insulin deficiency promotes it. In starvation, 50% protein synthesis is reduced [54] In experimental studies, proved that the protein
22
synthesis is considerably superior than the dose necessary to suppress proteolysis is depend on insulin dose promoting. Anabolic effect of insulin is conjunction with IGF-1, growth hormone, and enough amino acids. [54] Muscle glycogen synthesis is impaired in insulin resistance; this appears mainly mediated by condensed intracellular glucose translocation. [49]
Liver increased free fatty acid flux tends to support hepatic very low density lipoprotein (VLDL) assembly whilst ketogenesis typically remains suppressed by the compensatory hyperinsulinaemia. In insulin resistance the effects on adipose tissue are similar. Furthermore, insulin resistance impairment and lipoprotein lipase action is insulin-dependent, peripheral uptake of triglycerides from VLDL is also diminished. These mechanisms may contribute to the observed hypertriglyceridaemia of insulin resistance.38 In addition to free fatty acids, adipose tissue secretes a amount of cytokines which have systemic effects on insulin resistance such as IL-6, TNFα, angiotensinogen and leptin which are associated with increased insulin resistance, and adiponectin with insulin resistance reduction. [55] TNFα and IL-6 impairs insulin signalling, endothelial function and lipolysis. IL-6 assembly is improved by sympathetic nervous system activation, e.g. stress. [55] Adipose tissue depots differ in insulin response. [54] GLUT 4 translocation reduced in adipocytes from diabetic and insulin resistant individuals, impaired intracellular signalling via reduced IRS-1 gene and protein expression. [53]
23 Liver
Liver glucose uptake is not insulin-dependent, it accounts for about 30%
of whole body insulin-mediated glucose removal, [53] with insulin being needed to facilitate key metabolic processes., glycogen synthesis is stimulated while protein synthesis and lipoprotein metabolism are modulated through intracellular signalling which was described above.30 Gluconeogenesis and ketone body production are inhibited. Hepatic production of insulin-like growth factors and potentially mediated by mitogenic effects of insulin (and growth hormone) via suppression of sex-hormone binding globulin (SHBG) production. [49]
While in deficiency of insulin, e.g. starvation, these processes are more consistently affected; this is not unavoidably the case with insulin resistance.
Compensatory hyperinsulinaemia, discrepancy insulin resistance and differential tissue needs may dissociate these processes. [51] Insulin’s resistance metabolic effects results in increased output of glucose via increased gluconeogenesis (as in starvation), however, unlike starvation, compensatory hyperinsulinaemia depresses sex hormone binding globulin production and promotes mitogenic effects of insulin. Alterations in lipoprotein metabolism correspond to a major hepatic demonstration of insulin resistance. Increased free fatty acid liberation, and reduced catabolism of very low density lipoprotein by adipocytes of insulin resistant, results in increased hepatic triglyceride content and secretion of very low density lipoprotein. [56] Hepatic synthesis of C-reactive protein and fibrinogen is induced in response to
24
adipocyte-derived pro-inflammatory cytokines such as TNFα and IL-6. Gene expression of factor VII may also increased by insulin. [55]
SYNDROME OF INSULIN RESISTANCE
The syndrome of insulin resistance describes the cluster of abnormalities which occur more frequently in individuals in insulin resistant. These include glucose intolerance, endothelial dysfunction, dyslipidaemia, and increased pro- coagulant factors, changes in haemodynamics, enhanced inflammatory markers, abnormal metabolism of uric acid, elevated ovarian testosterone secretion and sleep-disordered. [51] Type 2 diabetes is the major clinical syndromes associated with insulin resistance include, cardiovascular disease, hypertension, syndrome of polycystic ovary, fatty liver disease of non- alcoholics, certain forms of cancer and sleep apnoea. [51]
INSULIN RESISTANCE ASSOCIATED CONDITION Type 2 Diabetes
Bornstein[57] and the Nobel Prize-winning effort of Yalow and Berson,
[58] the first insulin assays became extensively available in the late 1960s; [49] it was later confirmed that diabetic patients with so-called or adulthood onset or type 2 diabetes had normal or elevated plasma insulin levels. Insulin resistance was reported to be a characteristic feature of T2DM in the early 1970s. [51] A progressive inability of the β cells to recompense for the widespread insulin resistance by enough hyperinsulinaemia, heralds the clinical beginning of this disorder. [51] While two studies and linkage analyses are reliable with a tough
25
genetic constituent in the improvement of type 2 diabetes, numerous decades of research have unsuccessful to recognize a predominant genetic deformity in the majority of cases. [59] The aetiology of T2DM is consideration to be polygenic, with ecological factors being superimposed upon this basic inclination.
Insulin resistance characteristically predates the augmentation of diabetes and is usually found in unchanged first-degree relatives. [49] The morbidity of the disorder relates both to the severity of hyperglycaemia and the metabolic penalty of insulin resistance itself. The primary defects in insulin action become visible to be in muscle cells and adipocytes, with impaired GLUT 4 translocation ensuing in impaired insulin-mediated glucose transport.
[49]
Compensatory hyperinsulinaemia develops primarily, but the first phase of insulin secretion is vanished early in the disorder. Additional environmental and physiological stresses such as weight gain, pregnancy, physical inactivity and medications may get worse in the insulin resistance. β cells fail to reimburse for the existing insulin resistance, impaired glucose tolerance and diabetes develops. As glucose levels rises, β cell function got deteriorates additionally, with diminishing sensitivity to glucose and decline hyperglycaemia. The pancreatic islet cell mass is reported to be condensed in size in diabetic patients; humoral and endocrine factors may be imperative in maintaining islet cell mass. [45] In contrast to large forms of type 2 diabetes, the genetic basis of Maturity Onset diabetes of the Young (MODY) has been fine characterised and relates to defects in glucokinase. [59]
26
INSULIN AND ITS RESISTANCE MEASUREMENT
There are a variety of approaches to the laboratory assessment of insulin resistance. Over the lifetime the partial specificity of older radio-immunoassays that cross-react with pro insulin has reduced the trustworthiness of measuring insulin resistance in medical settings. Present assays have enhanced specificity and precision. Insulin resistance may be calculated by looking directly at insulin mediated glucose uptake in the basal or post-stimulated condition, by inference from the relative concentrations of glucose and insulin, or by looking at surrogate markers of insulin action.
TYPES OF DM
There are two important types of diabetes, one is characterised by insulin deficiency, termed type 1 diabetes (T1DM), and the another one is, type 2 diabetes (T2DM), arises mainly due to insulin resistance secondary to increased prevalence of obesity. However, the two conditions may overlap as a significant proportion of individuals with T1DM develop a phenotype seen in T2DM, makes them fall into a new category termed as double diabetes. [39]
Equally, longer duration of T2DM can lead to insulin deficiency, making T2DM individuals similar to T1DM patients
FREQUENCY OF THYROID DISORDERS
Thyroid disorders are more common with variable prevalence among the different populations. Data from the Whickham survey, a study conducted in the late 1970s in the north of England showed a prevalence of 6.6% of
27
thyroid dysfunction in the adult general population. In the Colorado Thyroid Disease Prevalence study involving 25,862 participants attending a state health fair, 9.5% of the studied population were found to have an increased level of TSH, while 2.2% had a decreased level of TSH [60]. In the NHANES III study, a survey of 17,353 subjects representing the US population, hypothyroidism was seen in 4.6% and hyperthyroidism in 1.3% of subjects. Latter further observed an greater frequency of thyroid dysfunction with advancing age and a higher prevalence of thyroid disease in women when compared to men and in diabetic subjects compared to non-diabetic individuals . Many reports documented a higher prevalence of thyroid dysfunction in the diabetic population. Particularly, Perros et al. [60] demonstrated an overall prevalence of 13.4% of thyroid diseases seen in diabetics with the highest prevalence in type 1 female diabetic patients (31.4%) and lowest prevalence in type 2 male diabetic patients (6.9%).
Recently, a prevalence of 12.3% was reported that among Greek diabetic patients and 16% of Saudi patients with type 2 diabetes mellitus were found to have thyroid dysfunction. In Jordan a study described that thyroid dysfunction was present in 12.5% of type 2 diabetes mellitus patients.
However, thyroid disorders were found to be more commonly seen in subjects with type 1 diabetes compared to those with type 2 diabetes mellitus.
THYROID HORMONES ON GLUCOSE HOMEOSTASIS
Thyroid hormones have an effect on glucose metabolism via several mechanisms. Hyperthyroidism has long been recognized to encourage
28
increased blood glucose. During hyperthyroidism, the half-life of insulin is decreased most likely secondary to an increased rate of degradation and an intensified release of biologically inactive insulin precursors.
In elevated proinsulin levels in response to a meal were observed in a study by Bech et al. [61] In addition, untreated hyperthyroidism was affiliated with a reduced C-peptide to proinsulin ratio suggesting an fundamental defect in proinsulin processing. Another mechanism explaining that the relationship between hyperglycemia and hyperthyroidism is the increase in glucose gut absorption intervened by the surplus thyroid hormones.
Endogenous production of glucose is also increased in hyperthyroidism via several mechanisms. Thyroid hormones secretes an increase in the hepatocyte plasma membrane concentrations of GLUT2 which is the main glucose transporter in the liver, and accordingly, the increased levels of GLUT-2 contribute to the increased hepatic glucose output and unusual glucose metabolism.
Furthermore, an increased lipolysis is noticed in hyperthyroidism resulting in an elevation in FFA that trigger the hepatic gluconeogenesis. The increased release of FFA could relatively be explained by an strengthened catecholamine-stimulated lipolysis induced by the excess thyroid hormones [62]. Moreover, the nonoxidative glucose disposal in hyperthyroidism is enhanced resulting in an increased production of lactate that enters the Cori cycle and stimulates further hepatic gluconeogenesis. The increase in glucagon, growth
29
hormone, and catecholamine levels integrated with hyperthyroidism further contributes to the impaired glucose tolerance.
It is well known that diabetes mellitus patients with hyperthyroidism experience worsening of their glycemic control in blood and thyrotoxicosis has been prone to diabetic ketoacidosis in subjects with diabetes. In hypothyroidism, glucose metabolism is affected via several mechanisms [63, 64]. A inreased rate of liver glucose production is observed in hypothyroidism and it is a cause for the decrease in insulin requirement in hypothyroid diabetic patients.
MATERIALS AND METHODS
30
MATERIAL AND METHODS
A cross-sectional study was carried out during the period of February 2016 to June 2017 among all types were used for the investigation. Type 2 diabetes individuals on treatment for 3 years with age group of more than 30 to 50 years and the total number of subjects were taken for the study 100 samples attending the outpatient Department of Karpaga Vinayaga Institute of Medical Science for regular health checkup who have no known significant medical illness which can affect the outcome of the study.
This study was ethically approved by the institutional ethical committee. Age and Body mass index was quantified by bioelectrical impedance analysis. TSH, T4, T3, total cholesterol (TC), triglycerides (TGL), HDL-C, LDL-C, levels along with a written consent was taken from every patient. Fasting Serum sample from cases as well as control group was obtained to determine the following investigations. Thyroid function tests were measured by (Avantor Performance Materials, India) kit using enzyme linked Immunosorbent assay (ELISA). Normal range of thyroid tests was
TSH: 0.39–6.16 (µIU/ml)
Free T4: 0.8-2.0 (ng/dl) and FT3: 1.4 - 4.2 (pg/ml)
AVANTOR Kits were used for the investigation. Patients with TSH levels > 6.2 (µIU /ml) with normal FT4 & FT3 values were accepted to have SCH. Total cholesterol (130-250 mg/dl)triglycerides (60-170 mg/dl), HDL – cholesterol ( male:35-80mg/dl;female:42-88mg/dl )VLDL (20-40mg/dl), LDL (80-150 mg/dl). Blood sugar fasting (70-110 mg/dl) and post prandial (upto
31
130 mg/dl) and renal function test such as urea (15- 40 mg/dl), creatinine (male: 0.9-1.4 mg/dl; female 0.8-1.2 mg/dl) was investigated by GOD/POD method, enzymatic method respectively. Beacon kit were used for the investigation.HBA1C (5-7%) Quantia kit was used for investigation.
The subjects were studied in terms of their thyroid dysfunction and its risk factors among type 2 diabetes. The study parameter used was:
Assessment parameters: Lifestyle factors
Age (30-50 years)
Gender (males and females)
Physical activity and exercise wise: low, moderate, high
Socio ecnomic condition
Body mass index (BMI)
Alcohol intake
Blood pressure Exclusion criteria:
Type I diabetes mellitus individuals
Patients on medications affecting thyroid function
Total and hemi thyroidectomy individuals
Gestational diabetes mellitus
Cortico steroid therapy.
Diabetic keto acidosis.
Neurodegenerative diseases
Cerebrovascular diseases
32
Tuberculosis
Cancer.
Test for thyroid profile
HISTORY
History was elicited according to Pro forma enclosed in the annexure. Importance was given to their family history to look for history of drug history and menstrual history was taken while drug affecting thyroid function. History was the first level for the selection process of individuals to include them in the study.
GENERAL AND SYSTEMATIC EXAMINATION:
General examination including anthropometric measurements and systematic examination was done according to the proforma attached in the annexures.
BODY MASS INDEX CALCULATION:
Body mass index (BMI) was calculated by dividing weight (Kg) by height squared (m2)5.They were classified in terms of table
Table: WHO recommended BMI table for Asians
NORMAL 18.50-22.99
UNDER WEIGHT < 18.50
OVERWEIGHT 23-24.99
PRE OBESE 25-29.99
EXTREME OBESITY >30
33 ESTIMATION OF GLUCOSE
METHOD
GOD- POD (Glucose oxidase /peroxidise) Method (Trinder 1969)[65]
PRINCIPLE
Glucose is oxidised to gluconic acid and hydrogen peroxide in the presence of glucose oxidase. Hydrogen peroxide further reacts with phenol and 4- amino antipyrine by the catalytic action of peroxidises to form a red
coloured quinineamine dye. Intensity of colour is directly proportional to the concentration of glucose present in the sample.
REACTIONS
Glucose + O2 + H2O Glucose oxidase Gluconic acid + H2O2
H2O2 + 4-amino antipyrine + phenol peroxidise Red quinine imine dye + H2O
SAMPLES PREPARATION
Blood is collected in a tube containing heparin / sodium fluoride, mixed well and centrifuged . The separated plasma is used for analysis.
REAGENTS
Standard Glucose: 100 mg/dl Glucose reagent enzyme
34 PROCEDURE
Addition
Sequence Blank Standard Test
Glucose Enzyme
reagent 1 ml 1 ml 1 ml
Standard - 10µl -
Sample - - 10µl
Mix well and incubate at room temperature for 10 mins .
Measure Absorbance of the standard (Abs. S) and Absorbance of the test (Abs.T) against reagent blank at 505 nm
CALCULATION
Glucose conc. mg/dl = ( Abs. T ) / ( Abs.S ) × 100 NORMAL VALUE
Fasting Blood Glucose: 70-110mg/dl
Post Prandial Blood Glucose : Upto 130mg/dl
ESTIMATION OF UREA METHOD
Urease / Berthelot Method (Tietz 1986)[66]
PRINCIPLE
Urease breaks down urea into ammonia and carbon dioxide in alkaline medium, Ammonia liberated from the breakdown of Urea reacts with hypochlorite and salicylate to form dicarboxylindophenol. This reaction is catalysed by the presence of Nitropruside. The intensity of the colour produced
35
by the reaction is directly proportional to the concentration of urea present in the sample and it is measured photometrically at 600 nm (600-630 nm) .
Urea + H2O Urease 2NH3 + CO2
NH3 + Salicylate + CIO Sodium nitropruside 2, 2 dicarboxylindophenol
REAGENTS
Reagent 1 : Enzyme reagent Reagent 2 : Chromogen reagent Reagent 3 : Standard 40 mg/ dl
SAMPLES
Unhemolysed serum/ Heparinised plasma
REAGENT PREPARATION AND STABILITY Step 1: Bring all the reagents to Room temperature
Step 2: Working reagent 1 (W1 ) – Dissolve the enzyme reagent 1 in deionised water
Step 3 : Working reagent 2 (W2) – ready for use
Step 4 : Allow the reagents to stand for 5 minutes at R.T for equilibration Working reagents and Urea standard with value 40 mg/dl were provided in the kit and were ready for the assay and stable till their expiry date when stored at 2- 8C
36 PROCEDURE
Reagent Blank Standard Test
Working reagent 1ml 1ml 1ml
Standard - 10μl -
Sample - - 10μl
Mix well and incubate at 37 ̊ C for 5 minutes
Reagent 2 1 ml 1ml 1ml
Mix well and incubate at 37C for 5 minutes . Measure the absorbance of standard and sample at 600nm (580-630 nm)
CALCULATION
Urea .Conc.(mg/dl ) = Abs ( Sample) / Abs ( Standard ) × 40
REFERENCE VALUE Adults : 15- 40 mg/dl
ESTIMATION OF CREATININE [100-101]
METHOD
Alkaline picrate Method
PRINCIPLE
Picric acid in an alkaline medium reacts with creatinine to form an orange coloured complex with the alkaline picrate. Intensity of the colour formed is directly proportional to the amount of creatinine present in the sample.
37
Creatinine + Picric acid Alkaline medium Orange Coloured Complex
REAGENTS
Reagent 1 ; Creatinine Buffer reagent Reagent 2 : Creatinine Picrate reagent Reagent 3 : Creatinine Standard 2 mg/dl
SAMPLES
Unhemolysed serum
REAGENT PREPARATION AND STABILITY
Step 1 : Working reagent 1 is prepared by combining equal volumes of Reagent 1 and Reagent 2
Step 2 : Mix by gentle swirling
Step 3 : Allow the reagent mixture to stand at R.T for 5 minutes for equilibration
Working reagents and Creatinine standard with value 2mg/dl were provided in the kit and were ready for the assay and stable till the expiry date when stored at 2- 8C
PROCEDURE
Addition Sequence Standard Test
Working Reagent 1.0 ml 1.0ml
Standard 50μl -
Sample - 50μl
38
Mix well and read the initial absorbance (A) for the standard and Test after exactly 30 seconds. Read another absorbance (A) of standard and Test exactly 120 seconds later. Calculate the change in absorbance A for both the standard and Test
Determine for Standard AS = A2S – A1S For Test AT = A2T - A1T
CALCULATION
Creatinine Conc. mg/dl = AT / AS × 2
REFERENCE VALUE Serum :
Male : 0.9 – 1.4 mg/dl Female : 0.8 – 1.2 mg/dl
LIPID PROFILE
CHOLESTEROL ( CHOD / POD METHOD ) [102-104]
AIM
Quantitative estimation of Total Cholesterol in human serum by CHOD/POD method.
PRINCIPLE
Cholesterol esterase hydrolyses esterified cholesterols to free cholesterol. The free cholesterol is oxidised to form hydrogen peroxide which further reacts with phenol and 4-amino antipiyrine by the catalytic action of
39
peroxidise to form a red coloured quinine imine dye complex. Intensity of colour is directly proportional to the amount of cholesterol present in the sample.
REACTIONS
Cholesterol esters +H2o Cholesterol esterase Cholesterol + fatty acids Cholesterol + O2 Cholesterol oxidase Cholestenone + H2O2
H2O2 + Phenol + 4- amino antipyrine peroxidise Red quinineimine dye + H2O
REAGENTS
Reagent 1: Cholesterol enzyme reagent Reagent 2: Cholesterol standard 200 mg/dl Reagent 3: Cholesterol precipitating reagent
SAMPLES
Serum, Heparinised /EDTA plasma
REAGENT PREPARATION AND STABILITY
All reagents are ready to use and are stable till the expiry date , when stored at 2-8°C
PROCEDURE Addition
sequence Blank Standard Test
Enzyme reagent 1 ml 1 ml 1 ml
Standard - 10µl -
Sample - - 10µl
40 Mix well and incubate at 37°C for 5 mins.
Measure Absorbance of the standard (Abs . S ) and Absorbance of the test(Abs.T) against reagent blank at 505 nm
CALCULATION
Cholesterol mg/dl = Abs.T / Abs.S ×200
NORMAL VALUE
Serum : 130- 250 mg/dl
Interpretation of Results:
Increased Serum levels are seen in the following conditions:
Familial hypercholesteremia
Nephrotic syndrome
Biliary obstruction
Hypothyroidism
Pregnancy
Decreased Serum levels are seen in the following conditions:
Hyperthyroidism
Malnutrition
Chronic anemia
Thyroiditis
Severe liver insufficiency
41
TRIGLYCERIDES (GPO/POD METHOD) [105-107]
AIM
Quantitative estimation of triglycerides by enzymatic GPO - POD (glycerol phosphate oxidase – peroxidase) end point method. Screening the lipids of an individual is an important diagnostic feature to detect artherosclerotic risks. Triglycerides values are increased in primary and secondary hyperlipoproteinemias. It is also increased in conditions like Diabetes mellitus, nephrosis, biliary obstruction, and various metabolic abnormalities due to endocrine disturbances.
PRINCIPLE
Glycerol released from hydrolysis of triglycerides by lipoprotein lipase is converted by glycerol kinase into glycerol-3-phosphate which is oxidised by glycerol phosphate oxidase to dihydroxy acetone phosphate and hydrogen peroxide. In presence of peroxidise, hydrogen peroxide oxidises phenolic chromogen to a red coloured compound.
REACTIONS
Triglycerides LPG lipase Glycerol+ fatty acids
Triglycerides + ATP Glycerol kinase Glycerol- 3-P + ADP Glycerol-3-P +O2 Gly- 3- P oxidise DHAP+H2O2
H2O2 + Phenolic chromogen POD Red colour compound
42 REAGENTS
Reagent1: Triglycerides enzyme reagent Reagent 2: Triglycerides standard 200mg/dl
SAMPLES
Unhemolysed serum, heparinised plasma , EDTA Plasma
REAGENT PREPARATION AND STABILITY
All reagents are ready to use and are stable till the expiry date , when stored at 2-8°C
PROCEDURE Addition
sequence Blank Standard Test
Enzyme reagent 1 ml 1 ml 1 ml
Standard - 10µl -
Sample - - 10µl
Mix well and incubate at 37°C for 10 mins.
Measure Absorbance of the standard (Abs. S) and Absorbance of the test (Abs.T) against reagent blank at 505 nm
CALCULATION
Triglycerides mg/dl = ( Abs.T ) / (Abs.s ) ) × 200
NORMAL VALUE Serum : 60-170mg/dl
43 Interferences:
The criteria for no significant interference is recovery within 10% of the initial value.
Bilirubin: No significant interference up to 40 mg/dLBilirubin.
Hemolysis: No significant interference up to 500 mg/dLHemolysate.
Ascorbate: No significant interference up to 20 mg/dLAscorbate.
Calculation: Not Applicable.
Interpretation of Results:
Increased Serum levels are seen in the following conditions like Diabetes mellitus, Nephrotic syndrome, Pregnancy, Excessive alcohol intake, Familial hyper triglyceridemia, etc.,
Decreased Serum levels were seen in the following conditions like Malnutrition, Congenetial abetalipoprotenimia, etc.,
Potential Sources of Variability:
Presence of fibrin in the sample may lead to erroneous results and hence centrifuge the sample only after complete clot formation.
Lysed serum specimens may give falsely elevated values.
HDL CHOLESTEROL DIRECT REAGENT KIT [108-110]
AIM
Quantitative estimation of HDL – Cholesterol in human serum by Enzymatic colour test. Measurement of serum HDL – cholesterol is useful in the screening of the lipid status of the individual to detect atherosclerotic risks
